1. Field of the Invention
This invention pertains generally to vibrational spectroscopy, and more particularly to an apparatus and method for Raman spectroscopy including sample exposure to a chirped, pulsed probe beam and a Raman pump beam wherein temporal dispersion is employed to transform the Raman-scattered light into a time-domain waveform which is electronically detected and preferably converted to digital signals.
2. Description of Related Art
Raman spectroscopy is widely used to study and identify a variety of biological, mineral, and chemical species. It is a powerful tool because it can uniquely identify molecules in complex mixtures and chemical environments, allowing for molecular fingerprinting and determination of relative concentration. Raman spectroscopy is a type of vibrational spectroscopy that observes radiation called the Raman spectrum scattered from an applied beam of light called the pump.
It has been observed that when a light beam is directed on a sample, photons are either absorbed by the sample material or scattered. Most of the photons are elastically scattered and have the same energy, frequency, and wavelength as the incident photons. This process is known as Rayleigh scattering. However, a small fraction of the incident radiation is inelastically scattered from optical phonons and shifted to different wavelengths and frequencies. This process is called Raman Scattering. The majority of Raman scattered photons shift to wavelengths, called the Stokes wavelengths, which are longer than the incident wavelength. A minority of Raman scattered photons shift to wavelengths, called the anti-Stokes wavelengths, which are shorter than the incident wavelength. Stokes scattering is more intense than anti-Stokes scattering because the Boltzmann occupation factor of vibrational energy levels above the ground state is small.
The Raman effect occurs when an incident photon interacts with the electric dipole of a molecule resulting in an energy transition from the molecular vibrations. With Stokes scattering, a vibrational transition is induced when a photon strikes the molecule and is re-emitted at a longer wavelength because some of the original energy of the photon has been transferred to the molecule. In the case of anti-Stokes scattering, the photon takes up part of the vibrational energy of a molecule, which is already in a higher vibrational state, and is emitted at a shorter wavelength. Therefore, Raman scattering arises from changes in the vibrational energy of the target sample molecule. Although vibrational Raman scattering is the most commonly studied form, rotational and electronic Raman scattering are also possible. The vibrational energy levels of a molecule are dictated by the masses and other properties of the constituent atoms, bond strengths, hydrogen bonding and inter-molecular interactions, molecular geometry, and the chemical environment. The spacing between the vibrational energy levels of the sample molecule is equal to the difference between the energy of the incident photon and the Raman scattered photon.
A Raman spectrum is a plot of the intensity of the Raman scattered light as a function of the difference in energy (frequency) between the incident radiation and the Raman scattered radiation. The difference in frequency between the incident and Raman radiation is independent of the frequency of the incident radiation.
Accordingly, Raman interaction involves the coupling between pump (incident), Stokes, and anti-Stokes radiation mediated by optical photons in a Raman-active medium. In spontaneous Raman scattering, a pump beam with frequency ωP traveling through a Raman medium scatters off of atomic or molecular vibrations and/or rotations, which are oscillating at frequency ωV. This scattering process creates a small amount of optical radiation at the new frequencies ωS=ωP−ωV and ωaS=ωP+ωV representing the Stokes and anti-Stokes frequencies, respectively.
In the spontaneous situation, the amount of radiation scattered into the anti-Stokes mode is much smaller than that scattered into the Stokes mode because at equilibrium, most of the population occupies the lowest vibrational level. The Stokes radiation is also much weaker than the pump (typically their relative intensities are 10−6 if the interaction length is 1 cm.) However, the intensity of the scattered Stokes radiation can be greatly enhanced if the interaction is stimulated by some initial injection of radiation at the Stokes frequency. In this stimulated Raman scattering (SRS) process, a sizable fraction of the pump radiation can be converted to the Stokes mode. In the general case, the field amplitudes of the Stokes and anti-Stokes modes obey the following coupled differential equations:
                    ⅆ                  A          S                            ⅆ        z              =                            -                      α            S                          ⁢                  A          S                    +                        κ          S                ⁢                  A          aS          *                ⁢                  ⅇ                      ⅈ            ⁢                                                  ⁢            Δ            ⁢                                                  ⁢            kz                                                            ⅆ                      A            aS                                    ⅆ          z                    =                                    -                          α              aS                                ⁢                      A            aS                          +                              κ            aS                    ⁢                      A            S            *                    ⁢                      ⅇ                          ⅈ              ⁢                                                          ⁢              Δ              ⁢                                                          ⁢              kz                                            ,  
where AS and AaS are the slowly-varying amplitudes of the Stokes and anti-Stokes waves, αS and αaS are the gain/nonlinear absorption coefficients (on Raman resonance, αS<0 and αaS>0), κS and κaS are the coupling coefficients between the modes, and Δk is the wave vector mismatch between the pump, Stokes, and anti-Stokes waves. If Δk is large, phase matching does not occur and the Stokes and anti-Stokes amplitudes are decoupled from one another. In this case, the Stokes wave experiences gain, and the anti-Stokes wave experiences loss. This case is referred to as stimulated Raman scattering (SRS). If Δk=0, the modes are coupled and appreciable amounts of Stokes and anti-Stokes radiation can be created. In this situation, a Stokes seed causes radiation to be scattered into the anti-Stokes mode through coherent anti-Stokes Raman scattering (CARS) and an anti-Stokes seed causes radiation to be scattered into the Stokes mode through coherent Stokes Raman scattering (CSRS).
The typical Raman analysis instrument is composed of at least three parts: a light source, a scattered photon collector, and a spectrometer. The light source is typically a laser which generates a coherent beam of monochromatic light that can be used as a Raman pump. Furthermore, since laser radiation can be tightly focused onto a sample, a high degree of spatial resolution (i.e., microscopy) can be achieved in the analysis. The spatial resolution can be especially large for the intensity-dependent interactions of nonlinear Raman scattering because the interaction is strongest in the part of the beam which is most intense.
The scattered photons from the pump laser are collected by a photon collector. The collector typically may be designed to preferentially collect the Raman scattered light over the unwanted background signals, such as fluorescence. The collected Raman scatter is sent to the spectrometer. In conventional Raman spectroscopy (stimulated and spontaneous), the scattered light is typically analyzed by a grating-slit spectrometer. In this ubiquitous device, light is spatially separated into its spectral components by an optical grating and a slit scans across the separated components to determine the relative intensity of light at each frequency. The detector records the intensity of the Raman signal at each wavelength and plots the data as a Raman spectrum.
Unfortunately, since spectrometers usually require some type of mechanical scanning to sample the spectrum of the light, they have long acquisition times and require a continuous-wave or repetitive light source (i.e., they are unable to detect single ultra-fast pulses of optical radiation). Furthermore, the spectral resolution of the apparatus is typically related to the size of the spectrometer. Therefore, progressively larger, more expensive instruments are required to achieve greater spectral resolution, which prevents integration into microchip-scale electronics.
To perform fast acquisition, a multichannel array of photodetectors, often with post-detection amplifiers, may be used to simultaneously detect the entire spectrum. In this configuration, the system suffers from individual element mismatches, a problem that limits the dynamic range of the system. These mismatches are particularly difficult to calibrate when fast single-shot detection is desired. Specifically, the large RF bandwidth required of the detection circuitry renders calibration very difficult. The problem is somewhat similar to the interchannel mismatch problem which limits the dynamic range of multi-channel A/D converters.
Accordingly, there is an increasing need for high throughput Raman instruments for screening rare cell types, biomolecules, unknowns and chemical compounds with minimal sample preparation. There is a further need for miniaturized Raman spectrometers and instruments that have single-shot measurement capability. Such devices can be used, for example, in situations where continuous exposure to high intensity light could destroy the sample. There is also a need for a Raman spectrometer that is less sensitive to fluorescence that can normally mask the weaker Raman signals. The present invention meets these needs as well as others and is a significant improvement over the art.